Rieger syndrome (RS) is an autosomal dominant disorder of morphogenesis characterized by abnormalities of the anterior segment of the eye and dental hypoplasia. Other systemic anomalies have been reported in association with the syndrome, the most common of which are mild craniofacial dysmorphism, including maxillary hypoplasia and failure of involution of the periumbilical skin and omphalocele. Gastrointestinal defects reported include Meckels diverticulum (Krespi, Y. P. and D. Pertsemlidis (1979) Am. J. Gastroenter. 71:608-610) colon atresia (Rogers, R. C. (1988) Proc. Greenwood. Genet. Center 7:9-13) and anal stenosis (Crawford, R. D. (1967) Brit. J. Ophthalmol. 51:438-440) The ocular features can include a prominent, anteriorly displaced Schwalbe line, iris processes which insert into the Schwalbe line, hypoplasia of the anterior iris stroma or corectopiea and pseudopolycoria. Roughly one-half of the patients will develop glaucoma, usually by young adulthood. The dental features associated with the condition include oligodontia and microdontia.
There is general agreement that patients with both the ocular features and systemic abnormalities (dental, umbilical, craniofacial) should be designated Rieger syndrome. The nomenclature of patients with only the ocular features, however, is confusing and disputed. For example, the condition of only ocular abnormalities is sometimes referred to as Rieger syndrome and other times referred to as Axenfeld-Rieger anomaly.
The underlying genetic defect and the chromosomal localization of RS is uncertain. Cases with Axenfeld-Rieger anomaly have been reported with various aberrations of chromosomes 4, 6, 10, 13, 16, and 22 (Wilcox, L. M. et al., (1978) Am. J. Ophthalmol. 86:834-839; Tabbara, K. F. et al. (1973) Canad. J. Ophthal. 8:488-491; Herve, J. et al. 1984) Ann. Pediatr. 31:77-80; Stathacopoulas, R. A. et al. (1987) J. Ped. Ophthalmol. Strabismus 24:198-203; Akazawa, K. et al. (1981) J. Ophthalmol. 105:323; Furguson, J. G. and E. L. Hicks (1987) Arch. Ophthal. 105:323; Heinemann, M. et al. (1979) Br. J. Ophthalmol. 63:40-44). Four cases of fully developed RS, including the dental and umbilical anomalies, have been associated with deletions in the region of chromosome 4q23-26. One case of a deletion in the region of 4q26 which did not have RS has also been reported. These cases indicate that a gene for RS lies in the region of 4q25.
In RS, a high risk of developing increased intraocular pressures and subsequent glaucomatous damage necessitates vigorous screening procedures beginning in infancy. Current screening for the ocular abnormalities of RS requires slit lamp examination, tonometry, gonioscopy and a dilated examination of the optic nerve head.
The present invention is based on the discovery of novel molecules, referred to herein as RIEG nucleic acids and RIEG or xe2x80x9cSolurshinxe2x80x9d polypeptide molecules. The human RIEG gene, which is approximately 18 kb, consists of four exons, 222, 49, 206 and 1306 nucleotides in length. The ATG initiation codon is located in the second exon and the homeobox region in the third and fourth exons. Translation of the open reading frame yields a protein of about 271 amino acids.
The gene is expressed in oral epithelium, the umbilicus, and periocular mesoderm, consistent with the phenotypic abnormalities seen in Rieger syndrome patients. In addition, the gene is expressed in Rathke""s pouch and the vitelline artery.
In one aspect, the invention features isolated vertebrate RIEG nucleic acid molecules. The disclosed molecules can be non-coding, (e.g. probe, antisense or ribozyme molecules) or can encode a functional RIEG polypeptide (e.g. a polypeptide which specifically modulates, e.g., by acting as either an agonist or antagonist, at least one bioactivity of the human RIEG polypeptide). In one embodiment, the nucleic acids of the present invention can hybridize to a vertebrate RIEG gene or to the complement of a vertebrate RIEG gene. In a further embodiment, the claimed nucleic acid hybridizes with the coding sequence designated in at least one of SEQ ID Nos: 1 or 3 or to the complement to the coding sequence designated in at least one of SEQ ID Nos: 1 or 3. In a preferred embodiment, the hybridization is conducted under mildly stringent or stringent conditions.
In further embodiments, the nucleic acid molecule is an RIEG nucleic acid that is at least 70%, preferably 80%, more preferably 85%, and even more preferably at least 95% homologous in sequence to the nucleic acids shown as SEQ ID Nos: 1 or 3 or to the complement of the nucleic acids shown as SEQ ID Nos: 1 or 3. In another embodiment, the RIEG nucleic acid molecule encodes a polypeptide that is at least 90% and more preferably at least 94% similar in sequence to the polypeptide shown in SEQ ID No: 2.
The invention also provides probes and primers comprising substantially purified oligonucleotides, which correspond to a region of nucleotide sequence which hybridizes to at least 6 consecutive nucleotides of the sequences set forth as SEQ ID Nos: 1 or 3 or complements of the sequences set forth as SEQ ID Nos: 1 or 3, or naturally occurring mutants thereof. In preferred embodiments, the probe/primer further includes a label group attached thereto, which is capable of being detected.
For expression, the subject RIEG nucleic acids can include a transcriptional regulatory sequence, e.g. at least one of a transcriptional promoter (e.g., for constitutive expression or inducible expression) or transcriptional enhancer sequence, which regulatory sequence is operably linked to the RIEG gene sequence. Such regulatory sequences in conjunction with a RIEG nucleic acid molecule can be useful vectors for gene expression. This invention also describes host cells transfected with said expression vector whether prokaryotic or eukaryotic and in vitro (e.g. cell culture) and in vivo (e.g. transgenic) methods for producing RIEG proteins by employing said expression vectors.
In another aspect, the invention features isolated RIEG or Solurshin polypeptides, preferably substantially pure preparations e.g. of plasma purified or recombinantly produced RIEG polypeptides. In preferred embodiments, the polypeptide is a functional transcription factor.
In one embodiment, the polypeptide is identical to or similar to a RIEG protein represented in SEQ ID No: 2. Related members of the vertebrate and particularly the mammalian RIEG family are also within the scope of the invention. Preferably, a RIEG polypeptide has an amino acid sequence at least 60% homologous and preferably at least 80% homologous to the polypeptide represented in SEQ ID No: 2. The subject RIEG proteins also include modified protein, which are resistant to post-translation modification, as for example, due to mutations which alter modification sites (such as tyrosine, threonine, serine or aspargine residues), or which prevent glycosylation of the protein, or which prevent interaction of the protein with intracellular proteins involved in signal transduction.
The RIEG polypeptide can comprise a full length protein, such as represented in SEQ ID No: 2, or it can comprise a fragment corresponding to one or more particular motifs/domains, or to arbitrary sizes, e.g., at least 5, 10, 25, 50, 100, 150, 175, 200, 225, 250 or 260 amino acids in length.
Another aspect of the invention features chimeric molecules (e.g. fusion proteins) comprised of a RIEG protein. For instance, the RIEG protein can be provided as a recombinant fusion protein which includes a second polypeptide portion, e.g., a second polypeptide having an amino acid sequence unrelated (heterologous) to the RIEG polypeptide (e.g. the second polypeptide portion is glutathione-S-transferase, an enzymatic activity such as alkaline phosphatase or an epitope tag).
Yet another aspect of the present invention concerns an immunogen comprising a RIEG polypeptide in an immunogenic preparation, the immunogen being capable of eliciting an immune response specific for a RIEG polypeptide; e.g. a humoral response, an antibody response and/or cellular response. In preferred embodiments, the immunogen comprises an antigenic determinant, e.g. a unique determinant, from the protein represented in SEQ ID No: 2.
A still further aspect of the present invention features antibodies and antibody preparations specifically reactive with an epitope of the RIEG protein. In preferred embodiments the antibody specifically binds to an epitope represented in SEQ ID No: 2.
The invention also features transgenic non-human animals which include (and preferably express) a heterologous form of a RIEG gene described herein, or which misexpress an endogenous RIEG gene (e.g., an animal in which expression of one or more of the subject RIEG proteins is disrupted). Such a transgenic animal can serve as an animal model for studying cellular and tissue disorders comprising mutated or mis-expressed RIEG alleles or for use in drug screening. Alternatively, such a transgenic animal can be useful for expressing recombinant RIEG polypeptides.
In yet another aspect, the invention provides assays, e.g., for screening test compounds to identify inhibitors, or alternatively, potentiators, of an interaction between a RIEG protein and, for example, a virus, an extracellular ligand of the RIEG protein, or an intracellular protein which binds to the RIEG protein. An exemplary method includes the steps of (i) combining a RIEG polypeptide or bioactive fragments thereof, a RIEG target molecule (such as a RIEG ligand or a RIEG substrate), and a test compound, e.g., under conditions wherein, but for the test compound, the RIEG protein and target molecule are able to interact; and (ii) detecting the formation of a complex which includes the RIEG protein and the target polypeptide either by directly quantitating the complex, by measuring inductive effects of the RIEG protein, or, in the instance of a substrate, measuring the conversion to product. A statistically significant change, such as a decrease, in the interaction of the RIEG and target molecule in the presence of a test compound (relative to what is detected in the absence of the test compound) is indicative of a modulation (e.g., inhibition or potentiation of the interaction between the RIEG protein and the target molecule).
Yet another aspect of the present invention concerns a method for modulating the transcription of certain genes in a cell by modulating RIEG bioactivity, (e.g., by potentiating or disrupting RIEG bioactivity). In general, whether carried out in vivo, in vitro, or in situ, the method comprises treating the cell with an effective amount of a RIEG therapeutic so as to alter, relative to the cell in the absence of treatment, the level of transcription of certain genes Accordingly, the method can be carried out with RIEG therapeutics such as peptide and peptidomimetics or other molecules identified in the above-referenced drug screens which agonize or antagonize the effects of signaling from a RIEG protein or ligand binding of a RIEG protein. Other RIEG therapeutics include antisense constructs for inhibiting expression of RIEG proteins, and dominant negative mutants of RIEG proteins which competitively inhibit ligand interactions upstream and signal transduction downstream of the wild-type RIEG protein.
A further aspect of the present invention provides a method of determining if a subject is at risk for Rieger Syndrome or other disorder. The method includes detecting, in a tissue of the subject, the presence or absence of a genetic lesion characterized by at least one of (i) a mutation of a gene encoding a RIEG protein, e.g. represented in one of SEQ ID Nos: 1 or 3 or a homolog thereof; or (ii) the mis-expression of a RIEG gene. In preferred embodiments, detecting the genetic lesion includes ascertaining the existence of at least one of: a deletion of one or more nucleotides from a RIEG gene; an addition of one or more nucleotides to the gene, a substitution of one or more nucleotides of the gene, a gross chromosomal rearrangement of the gene; an alteration in the level of a messenger RNA transcript of the gene; the presence of a non-wild type splicing pattern of a messenger RNA transcript of the gene; a non-wild type level of the protein; and/or an aberrant level of soluble RIEG protein.
For example, detecting the genetic lesion can include (i) providing a probe/primer comprised of an oligonucleotide which hybridizes to a sense or antisense sequence of a RIEG gene or naturally occurring mutants thereof, or 5xe2x80x2 or 3xe2x80x2 flanking sequences naturally associated with the RIEG gene; (ii) contacting the probe/primer to an appropriate nucleic acid containing sample; and (iii) detecting, by hybridization of the probe/primer to the nucleic acid, the presence or absence of the genetic lesion; e.g. wherein detecting the lesion comprises utilizing the probe/primer to determine the nucleotide sequence of the RIEG gene and, optionally, of the flanking nucleic acid sequences. For instance, the primer can be employed in a polymerase chain reaction (PCR) or in a ligation chain reaction (LCR). In alternate embodiments, the level of a RIEG protein is detected in an immunoassay using an antibody which is specifically immunoreactive with the RIEG protein.
Other features and advantages of the invention will be apparent from the following detailed description and claims.